Formulation for promoting targeted pollination of pear tree crops in honey bees

ABSTRACT

A formulation and a composition that comprises it for promoting the pollination of pear crops (Pyrus) by biasing the foraging preferences of the honey bee (Apis mellifera). The formulation comprises the compounds limonene, linalool and α-pinene. Additionally, a method for targeting the bees&#39; pollination activity towards the pear crops by using the formulation comprising the compounds limonene, linalool and α-pinene.

The present disclosure relates to a formulation and compound thatpromote the pollination of pear tree crops (Pyrus sp.) by biasing theforaging preferences of the honey bee (Apis mellifera). It also relatesto a method for targeting the pollination activity of the bees towardspear tree crops.

BACKGROUND OF THE INVENTION

Among honey bees, odors play an important role in the search andselection of their nourishment sources. It is widely known that beescontrol the collection of resources not only according to innate searchimages, but also according to experiences previously acquired in thefield as well as in the social environment of the hive (Ribbands C R(1955) The scent language of honey bees; Ann Rev Smithson Inst 368-377;von Frisch K (1967) The dance language and orientation of bees; HarvardUniversity Press. Cambridge, Mass.). In this sense, the olfactorymemories established in the field based on floral odors and their nectarmay be retrieved (evoked) with the presence of this floral scent insidethe hive (Ribbands C R (1954) Communication between honeybees: theresponse of crop-attached bees to the scent of their crop; Proc REntomol Soc Lond A 29:141-144; Johnson D L & Wenner A M (1966) Arelationship between conditioning and communication in honeybees; AnimBehav 14:261-265; Jakobsen H B & col., (1995) Can social bees beinfluenced to choose a specific feeding station by adding the scent ofthe station to the hive air? J Chem Ecol 21(11):1635-1648; Reinhard J &col., (2004) Floral scents induce recall of navigational and visualmemories in honeybees. J Exp Biol 207:4371-4381). Furthermore, theconsumption of scented food retrieved by foragers and brought into thehive can produce a bias in the collecting preferences of their nestmates(von Frisch K (1923) Über die Sprache der Bienen; Zool Jb Physiol40:1-186; Wenner A M & col. (1969) Honey bee recruitment to foodsources: olfaction or language? Science 164:84-86; Dornhaus A, Chittka L(1999) Evolutionary origins of bee dances; Nature 401:38).

During foraging in scented feeding stations, hive members may learnthese odors if they remain impregnated in the foragers' bodies (vonFrisch K (1967) The dance language and orientation of bees; HarvardUniversity) and/or if small samples of scented food are transferredmouth-to-mouth between hive members (trophallaxis) (Farina, W M & col.(2007) Honeybees learn floral odors while receiving nectar from foragerswithin the hive; Naturwissenschaften, 94:55-60).

Furthermore, it has been demonstrated that scented food retrieved byforagers and moved around the hive may affect the behavior of young beesthat are not yet involved in foraging tasks (Goyret J & Farina W M(2005) Non-random nectar unloading interactions between foragers andtheir receivers in the honeybee hive. Naturwissenschaften, 92: 440-443;Grüter C & col. (2006) Propagation of olfactory information within thehoneybee hive. Behav Ecol Sociobiol 60:707-71; Grüter C & col. (2009).Retention of long-term memories in different age-groups of honeybee(Apis mellifera) workers. Insectes Sociaux, DOI10.10071s00040-009-0034-0).

The inventors of the present disclosure have previously publishedresults suggesting that bees may develop olfactory memories inside thehive during the distribution of scented food. Such memories may beretrieved (evoked) days after the fact and outside the hive, which isevidenced by the bias in the foraging preferences. The establishment ofthese memories depends on neither the recruiting mechanisms commonlyfound in hives, nor on the presence of scented food reserves that enablethe retrieval of the previously acquired memories (Arenas A & col.,(2007). Floral odor learning within the hive affects honeybees' foragingdecisions. Naturwissenschaften, 94:218-222).

In “Floral odor learning within the hive affects honeybees' foragingdecisions”, published in Naturwissenschaften, 94:218-222, Arenas A &col., it is furthermore disclosed that the odor of food that isavailable “inside the beehive” produces long-term olfactory memoriesthat affect the behavior for selecting food sources outside the hive. Itis also disclosed that, in order to develop long-term memories of aspecific floral (pure) odor, such odor should be available whiledissolved in a sugar solution (sucrose solution 50% w/w), which can bemade available in a feeder located inside the hive (a common procedureused in apiculture). The pure odor should be diluted in the sugarsolution in minimal quantities (concentration: 50 μl of odor per 1 L ofsugar solution available).

In “Floral scents affect the distribution of hive bees around dancers”,Behavioral Ecology and Sociobiology (2007) 61:1589-1597, Jul. 4, 2007,Diaz, Paula C. & col., it is disclosed that the floral odor impregnatedin the body of a bee returning from a flower site attracts nestmates atthe time of engaging in the “dance of the bees”, a mechanism used bythis species to recruit other bees towards food sources inside the hive,besides from increasing the incidence of food samples transfer takingplace via mouth-to-mouth contact or trophallaxis.

Lastly, in “Floral scents experienced within the colony affect long-termforaging preferences in honeybees”, Apidologie 39:714-722, 2008, ArenasA & col., it is disclosed that the factor increasing the number oflandings on a food source with a specific odor is the odor of the foodmoving around the hive and not the exposure to that same odorant asvolatile. Furthermore, in “Passive volatile exposure within the honeybeehive and its effect on odor discrimination”, Journal of ComparativePhysiology A, 195:759-768, 2009, Fernández V & col., it is disclosedthat the exposure to a floral odor as volatile “inside the hive” hinderslearning of those floral odors in subsequent trainings. This suggeststhat those odors exposed as volatilized compounds inside the hive willnot be preferred if they are present in a natural floral fragrance, evenwhen specific flowers offer abundant nectar as resource.

Many crops require pollination by insects that favor cross-fertilizationby visiting different flowers of the same species. Particularly, Apismellifera is the most abundant pollinator in single-crop farming aroundthe world, and is considered to be of very high importance to increasethe yield of seed and fruit.

In some countries leading the world ranking in honey production, as isthe case of Argentina, apiculture is essentially understood as a meansto obtain the produce stockpiled in the beehive (honey, propolis or beeglue, and royal jelly, etc.). Nonetheless, the use of the honey bee as acrop pollinizing agent is generally somewhat underdeveloped incomparison with other countries where it enjoys a greater importance,i.e. the United States and France. Nevertheless, in parallel with theadvances in monocultures or single-crop farming, a growing demand arisesin search of increasing targeted and sustainable pollination servicesfor these crops.

The main problem to be addressed in targeted pollination relates to thechange in floral availability when displacing hives from one environmentwhich is familiar to the foragers to another, unfamiliar location. In anunfamiliar environment, honey bees lack any reliable and updatedinformation that can be evoked during foraging.

In practice, it is observed that after transhumance (beehivedisplacement) the foragers remain relatively idle during the first days,and do not immediately visit the desired flowers, even when these offersubstantial rewards. This occurs due to the fact that the recentlyintroduced bees lack previous experiences related to the target crop;thus, nor previously established memories exist to guide or enablelocalization of this floral species. During a variable period (that cancarry on for several days) the bees develop new scent-nectarassociations (memories) and update the information that allows them tolocate new floral species available in the environment.

Two products available in the market, POLLINUS® and BEE SCENT®, aresimilar to a bee pheromone and their presence on floral sites attractsnew honey bees. Their use involves spraying of a crop (whatever itstype) with the product, which can be efficient in reduced spaces (suchas little orchards and greenhouses) but not over wide field surfaces.This would require several product sprayings, which would render thepractice overly costly and hardly efficient for extensive crops. Whilethese products act directly as bee attractants (although they might aswell attract other insects), they lack specificity for a particularfloral species, and due to their nature, they could interfere directlywith other behaviors of the bees. None of these products is based onfloral odors linked to appetite nor do they involve the bees' olfactorymemory.

Patent application CN102823628 (A) discloses a composition to attractbees to soy crops and its method of use, wherein the attractingcomposition is sprayed over the soy crop to promote its pollination, andtherefore increase production.

An alternative strategy is to “train” the bees so that they acquire apreference to collect pollen from a particular species of flower, inorder to promote its pollination. This strategy was implemented in the'30s by beekeepers of Germany and ex-USSR countries, who used thismethod to stimulate beehives with natural scents, particularly bygrinding flowers [(von Frisch, K. (1943), Versuche über die Lenkung desBienenfluges durch Duftstoffe. Naturwissenschaften 31, 445-460]. Thedisadvantage of this methodology is that industrial scale production ofscents via this procedure is not efficient, and requires the destructionof flowers belonging to the target species. Moreover, scents producedvia the grinding of flowers are unstable and do not easily resiststorage.

Patent application CN101569286 (A) discloses compositions and methods topromote pollination by bees of sterile plant parental lines. Thecomposition comprises secondary metabolites of flowers such as commonalkaloids (i.e. nicotine and caffeine) and water-soluble phenols (i.e.flavonoids, quercetin, gallic acid and caffeic acid). The methodconsists of placing the beehives inside tents where target plants grow.The bees are then fed with the composition that comprises the secondarymetabolites in a certain timeline, and feeding continues until floweringstage ends, at which point the bees are removed from the tent. Thismethod and composition are not targeted to any particular plant species,and are not useful for crops in large-scale farming, such as the peartree.

Patent application JP2008212148 “Method for promoting pollination ofplant including induction of flower bee to floral organ of specificplant by taking advantage of floral fragrance component of flower organof this plant” describes a composition of floral fragrances to promotepollination of plants by bees. Nonetheless, this patent application doesnot describe a composition that simulates the pear tree floral odor in aspecific manner, nor that is useful to target the bees' pollinationactivity towards pear tree crops. Neither can a compound of suchcharacteristics be derived from the disclosures of applicationJP2008212148.

Patent application WO2013005200 (A1) discloses a composition to promotepollination of apple crops (Malus silvestris) by causing a bias in thehoney bees' foraging preferences, and its method of use. The compositioncomprises the compounds citral, α-pinene and limonene. By being specificto apple crops, the composition is not useful to promote pollination ofpear tree crops.

Patent application WO2013005199 (A1) discloses a composition to promotepollination of sunflower crops (Helianthus annuus) by causing a bias inthe honey bees' foraging preferences. The composition comprises thecompounds sabinene, β-pinene and limonene. By being specific tosunflower crops, the composition is not useful to promote pollination ofpear tree crops.

Therefore, the need arises for effective and stable compositions thatpromote pollination of pear tree crops, and can also be produced in asimple and cost-effective manner, on an industrial scale, as well asmethods for promoting the pollination of pear tree crops by the honeybee (Apis mellifera).

BRIEF DESCRIPTION OF THE INVENTION

It is therefore an object of the present disclosure to providecompositions that simulate the pear tree floral odor in order togenerate specific olfactory memories in bees from hives that shall belocated in pear tree crops to promote their pollination. The memoriesestablished after stimulation with the present formulation decrease thetime during which bees remain idle until the beginning of foraging. Thecomposition of the invention stimulates collection of resources bypromoting a faster and constant foraging activity on the crop. The useof the invention decreases the time needed for the beehives to remain inthe crops, as well as the number of required beehives. Additionally, theuse of the composition improves the health conditions in the beehivesand increases their population. On the other hand, the composition ofthe invention has a very low cost and does not require the use ofsophisticated techniques for its implementation.

According to a first aspect, the present invention relates to aformulation for promoting the targeted pollination of pear tree crops byhoney bees, comprising the compounds limonene, linalool and α-pinene.

In one particular embodiment, the formulation of the invention comprises21.6% to 26.4% limonene, 31.5% to 38.5% linalool, and 36.9% to 45.6%α-pinene, and more particularly 24% limonene, 35% linalool and 41% deα-pinene.

According to another aspect, the present invention provides acomposition for promoting the targeted pollination of pear tree crops byhoney bees, comprising the formulation of the invention diluted in asugar solution.

In a preferred embodiment of the composition of the invention, the sugarsolution is an aqueous sucrose solution, more preferably a 50% w/wsucrose solution. In a more specific embodiment, the composition of theinvention comprises 0.1 ml to 0.2 ml of the formulation according to theinvention per 1 L of 50% w/w aqueous sucrose solution. In an evenfurther specific embodiment, the composition of the invention comprises0.1 ml to 0.2 ml of a formulation comprising 24% limonene, 35% linalooland 41% α-pinene per 1 L of 50% w/w aqueous sucrose solution.

According to another further aspect, the present invention provides amethod for promoting the targeted pollination of pear crops by honeybees, comprising the following steps:

a) administering the composition of the invention to the beehives;

b) keeping the beehives in or in the immediate vicinity of the pear treecrops, whose pollination can be promoted until the end of flowering; and

c) removing the beehives.

According to a particular embodiment of the method of the invention,step a) is executed via an artificial feeder located inside the beehive.

According to another particular embodiment of the method of theinvention, step a) takes place within 2 days before placing the beehivesin the crops, the pollination of which is sought to be promoted.

According to an additional particular embodiment of the method of theinvention, step a) is executed after the placement of the beehives inthe crops, the pollination of which is sought to be promoted.

According to an additional particular embodiment of the method of theinvention, step a) is executed before the crop's flowering level reaches20%.

According to an additional particular embodiment of the method of theinvention, the composition is administered to the beehives a second timeduring step b).

DESCRIPTION OF THE FIGURES

FIG. 1. Bees' ability to differentiate the pear tree natural floral odorfrom different scents in the proboscis extension response (PER) setup.A-C: Bees' behavior upon exposure to the pear tree natural floral odorvs.: A, Formulation I; B, Formulation II; C, the Formulation of theInvention (Formulation). The left panel shows the acquisition curvesduring training, while the right panel shows later evaluations. The sizeof the sample is indicated in the left panel. Asterisks indicatesignificant differences, ***, p<0.001; ** p<0.01; n/s, p>0.05 (Fisher'sexact test).

FIG. 2. Bees' ability to differentiate after Absolute OlfactoryConditioning of the proboscis extension response (PER) using theformulation of the invention (Specific Formulation). The left panelshows the acquisition curve during training, measured as the percentageof bees that extended their proboscis (% PER). The right panel shows thetests after the training. The sample size is indicated in the leftpanel. During the Test, three odors were presented without a reward: thepear flower, the apple flower and the Pear Specific Formulation of theinvention (SF). The different letters over the bars indicate significantdifferences with p<0.001 (G Test).

FIG. 3. Effect of the Pear Specific Formulation of the invention on theamount of collected pollen. The figure shows the total pollen collectedby bees of different beehives subjected to two treatments during theflowering stage: i) Control Group, beehives fed with 500 ml of SucroseSolution (white circle) in a single dose; ii) Treatment Group, beehivesfed with 500 ml of Sucrose Solution with the addition of the Pear treeFormulation of the invention (Specific Formulation, black circle). A)Percentage of the crop's flowering stage level during the test. B)Weight of 10 corbiculae with pear pollen (mg, median±ES) entering thehives during the flowering period (N=20 weight measurements pertreatment). The dotted line indicates the moment the treatments wereapplied. Asterisks indicate significant differences, **, p<0.01, ***,p<0.001 (ANOVA of iterated measurements). C) Pollen loads stored intraps placed in the entrance to the beehives (1 hive per treatment). Thesamples were classified in pear pollen (black bars) and pollen fromother species (white bars). The accrued values during 3 days afterapplying the treatments are shown. Asterisks indicate significantdifferences, ***, p<0.001 (Chi-squared Homogeneity Test).

FIG. 4. Brood area growth in beehives subjected to two treatments: i)Control Group, hives fed with 500 ml of Sucrose Solution (white bar, SS)in a single dose; ii) Treatment Group, hives fed with 500 ml of SucroseSolution with the addition of the Pear tree Specific Formulation of theinvention (black bar, EF). The hives' total brood area was estimatedbased on the sum of the brood areas of both sides in every frame. Thenumber of hives is indicated in brackets. The difference between theinitial brood area (measured the day before stimulation) and the finalbrood area (measured 7 days after) is shown. The asterisk indicatessignificant differences, *, p<0.05 (single factor ANOVA).

FIG. 5. Effect of the Pear Specific Formulation of the invention on thecrop yield. A. Crop's Yield measured in the direct vicinity of beehivessubjected to two treatments: i) Control Group, hives fed with 500 mlSucrose Solution (white) in a single dose; ii) Treatment Group, hivesfed with 500 ml Sucrose Solution with the addition of the Pear SpecificFormulation of the invention (black). The main graph shows the number offruits per tree, being the number of trees within an environment of 30 mshown in brackets. B. Number of fruits per tree analyzed in relation tothe number of beehives, comparing trees in the vicinity of untreatedhives with trees in the vicinity of hives treated with the SpecificFormulation. Asterisks indicate significant differences, *, p<0.05, **,p<0.01 (single factor ANOVA). The percentages indicate the increase inyield of the treatment group.

DETAILED DESCRIPTION OF THE INVENTION

In recent years, pollination of pear crops using honey bee hives hasgained significant relevance due to the greater increases in theresulting fruit yield.

The presence of honey bee hives in the vicinity of pear trees since thebeginning of the flowering stage causes a greater number of fertilizedflowers, and therefore a greater fruit setting, which in turn derived ina greater number of harvested fruits. Furthermore, the resulting fruitsare greater in size and containing a higher number of seeds, parameterslinked with a better quality of the pears of different varieties thatare exported as superior-level produce around the world.

For these reasons, pear producers hire pollination services fromapiarists specialized in beehive transhumance, renting a certain numberof hives during blooming period. In this context, and due to the factthat apiarists usually stimulate their hives with sugar solutions, theapplication of a composition containing an pear floral syntheticformulation does not represent a significant modification in the usualbeekeeping practices. When beekeepers relocate their hives during thisperiod, bees from these colonies show delays before starting to visitnovel flowers. As a consequence, being able to develop “memories of asynthetic formulation that simulates the pear fragrance” inside thebeehives would enable a reduction in such delay besides from makingpollination more efficient.

It is therefore an object of the present invention to provide aformulation that simulates the pear natural floral odor in order tocause bees to immediately start foraging on pear crops. Such formulationcomprises the compounds limonene, linalool and α-pinene. Particularly,this formulation acts by increasing the bees' foraging activity,attracting them specifically to pear flowers, promoting pollination and,as a result, increasing the crops' yield.

The inventors have further determined the optimal proportions for thecomponents of the formulation of the invention. Nonetheless, the expertin the field shall note that these values may vary within certainmargins without decreasing their efficiency, preferably in approximately+1-10% of their relative percentage. Particularly, the formulation ofthe invention that that simulates the pear tree floral odor comprises21.6% to 26.4% limonene, 31.5% to 38.5% linalool, and 36.9 to 45.6%α-pinene. Preferably, the formulation of the invention that thatsimulates the pear tree floral odor comprises 24% limonene, 35%linalool, and 41% α-pinene. Additionally, according to the presentapplication the terms “limonene”, “linalool” and “α-pinene” comprisesaid compounds per se as well as their derivatives, polymorphs,hydrates, solvates, enantiomers, etc.

Surprisingly, the inventors have found that although the formulation ofthe invention combines only three out of the 24 volatile compoundsidentified in the pear tree floral extract (Baraldi et al. 1999.Volatile organic compound emissions from flowers of the most occurringand economically important species of fruit trees. Phys. Chem. Earth B.24, 6, 729-732; see Example 1), bees cannot differentiate the scent ofthe natural flower from the formulation of the invention, even thoughthe relative abundance of each of the compounds in the formulation ofthe invention differ significantly from their relative percentage in thepear natural floral fragrance.

Even more surprisingly, the formulation of the invention shows a betterperformance than other volatile compounds combination present in thepear flowers that were expected a priori to show better results. Infact, as demonstrated in Example 2, the formulation of the invention hasa better performance than similar formulations.

Additionally, the field tests show as well that beehives fed with acomposition comprising the formulation of the invention, which is alsoan object of the present application, begin foraging activity earlierand in a more prolonged manner than control hives.

According to the method of the invention, the formulate of the inventioncan be added to the beehive before or during the hive's placement in thepear crops, using common methodologies already known to apiarists. Theinvention's formulation can be introduced in the beehive as part of acomposition, which is also an object of the present application, whereinthe formulation is diluted in a concentration of 0.1 to 0.2 ml per 1 Lof 50% w/w aqueous sugar solution (e.g. sucrose solution). Even morepreferably, the composition of the invention comprises 0.1 to 0.2 ml ofa formulation comprising 24% limonene, 35% linalool and 41% α-pinene,diluted in a 50% w/w sucrose solution. The composition of the inventionpromotes pollination of pear crops by honey bees.

For the purposes of the present invention, the abbreviation “w/w” refersto the relation between the solute weight and the total solution weight.

As to the way of administering it to the beehive, the formulation (i.e.as a part of a composition as described) can be placed via an artificialfeeder inside the hive, or directly by pouring 500 to 1000 ml of thesugar composition over the beehive frames. Eventually, a secondadministration can be done depending on the state of the beehives andthe crops, particularly when the blooming is very prolonged.

The addition of the formulation of the invention, combined with sugarsyrup before or during the placement of the beehives in the pear treecrops promotes quick and sustained foraging activity over the targetcrop, as well as an increased in foraging activity. This simplestimulation method enables the pollination of the pear crop and requiresless time for pollination and a smaller number of beehives to be used.On the other hand, the formulation of the invention has a low productionand commercialization costs.

In contrast to commercial products POLLINUS® and BEE SCENT®, whichgenerate innate responses in the behavior of the bees (which can becounterproductive in many cases), the formulation of the invention doesnot such “rigid” responses over the bee's behavior. On the contrary, itinfluences over the decision-making processes that are adaptable and canbe adjusted according to the needs of each beehive or to the changingenvironment conditions. This proves to be essential when attempting tocarry out integrated apicultural and agricultural management withoutdamage for any of the parties involved.

Therefore, the formulation of the invention can be used in small amountsto stimulate the beehives. Moreover, it is a simple formulation,comprising only three of the many volatile compounds that constitute thepear tree floral fragrance, resulting in a cost-effective product, whichis simple to manufacture and easy to use. Moreover, the formulationstimulates the immediate foraging of honey bees on pear crops.Stimulation via administering the formulation or the composition of theinvention can take place before taking the beehives to the crop or evenbefore the beginning of the pear blooming.

EXAMPLES Example 1: Preparation of the Formulations

In order to obtain the pear natural floral fragrance used in thefollowing Examples, pear or apple (respectively) buds (flowers that areclose to opening their petals or have very recently opened them) wereplaced in a clean glass container with two orifices that allow thegeneration of an air current to drag the flowers' volatiles. Saidcurrent is delivered to the bees' antennae and is used as a conditioned,non-rewarded stimulus, conditioned rewarded stimulus or testing odor.

3 different formulations were prepared, in each of them the 3 presentvolatile compounds in the pear tree flower were combined, identified byBaraldi et al. 1999 (Phys. Chem. Earth B. 24, 6, 729-732). Thecomposition of each of the formulations was the following:

-   -   Formulation I: 15% 2-ethylhexanol, 55% linalool, 30% limonene.    -   Formulation II: 24% 2-ethylhexanol, 35% linalool, 41% α-pinene.    -   Formulation III: 24% limonene, 35% linalool, 41% α-pinene.

Example 2: Testing of the Formulations Using Olfactive Conditioning

An absolute olfactory conditioning was executed to evaluate the bees'ability to differentiate the pear tree natural floral odor from otherscents. Each bee used in the trial was tied to a harness and presentedwith the scent in evaluation linked to a reward (Reward-conditionedStimulus, EC+), and were also presented with the pear tree naturalfloral odor without a reward (Non Reward-conditioned Stimulus, EC−). Theodors were presented in a pseudo-random order. The evaluated scents(EC+) were: A) Formulation I from Example 1 (n=29), B) Formulation IIfrom Example 1 (n=30), and C) Formulation III (Specific Formulation)from Example 1 (n=49). In all cases, non-rewarded control (EC−) was thepear tree natural floral odor obtained as described in Example 1. Theability to differentiate EC+ from EC− was evaluated based on the bees'proboscis extension response (PER).

By the end of the conditioning, the bees were able to differentiate thepear natural flower from Formulation I and II, but were unable todifferentiate the pear tree natural floral odor from Formulation III(Specific Formulation; FIG. 1).

Example 3: Evaluation of Formulations Through Differential OlfactoryConditioning

An absolute olfactory conditioning was executed to evaluate the bees'ability to differentiate the pear natural floral odor from the applefloral odor and Formulation III (Specific Formulation). Harnessed bees(n=30) were trained by being presented with Formulation III (SpecificFormulation) of example 1 linked to a reward. By the end of theconditioning, the bees were presented with Formulation III (SpecificFormulation) and two unfamiliar scents: The apple natural floral odorand the pear natural floral odor, and the bees' proboscis extensionresponse (PER) was evaluated. As it happened in the experiment describedin Example 2, the bees conditioned with Formulation III (SpecificFormulation), after being presented with the pear tree natural floralodor showed a PER similar to that observed in the last presentation ofthe conditioning, indicating that they could not differentiateFormulation 3 from the pear natural floral odor, but were in fact ableto differentiate Formulation III (Specific Formulation) from the applenatural floral odor (FIG. 2).

Example 4: Beehive Stimulation

A composition was prepared by dissolving 0.1 ml of Formulation 3 fromExample 2 per 1000 ml of an aqueous sucrose solution 50% w/w. Theformulation of the invention diluted in 500 ml of sucrose solution wasfed to 20 beehives through an internal plastic feeder placed insidethem. Meanwhile, other 20 hives received 500 ml of non-scented 50% w/wsucrose solution (control) through an internal feeder. In all cases, asingle administering was carried out per flowering stage. The hives werelocated in a pear tree (Pyrus) crop not far from General Roca (Rio NegroProvince, Argentina). The amount of collected pollen and the hives'population increase were measured. The crops' yield was also evaluatedin terms of fruits per tree.

The bees from hives fed with the solution scented with Formulation III(Specific Formulation) showed a higher weight of pollen load (FIG. 3B),and a greater amount of pollen loads, explained mainly by a greateramount of pear tree pollen loads in comparison with pollen loads ofother species (FIG. 3C).

The administering of the composition containing Formulation III(Specific Formulation) also resulted in a greater hives' populationincrease (FIG. 4), showing that the sugar composition with FormulationIII (Specific Formulation) promotes a greater food foraging andstimulates hive growth.

Additionally, beneficial effects in the pear tree crops were confirmed.The pear three crops in the vicinity of the beehives treated with thesugar composition with Formulation III (Specific Formulation) had ahigher number of fruits per tree (FIG. 5A). This higher yield is evenmore evident when the number of fruits per tree was analyzed in relationto the number of hives (FIG. 5B).

1. A formulation that promotes targeted pollination of pear crops byhoney bees, wherein the formulation comprises the compounds limonene,linalool and α-pinene.
 2. The formulation according to claim 1, whereinthe formulation comprises 21.6% to 26.4% limonene, 31.5% to 38.5%linalool and 36.9% to 45.6% α-pinene.
 3. The formulation according toclaim 2, wherein the formulation comprises 24% limonene, 35% linalooland 41% α-pinene.
 4. A composition that promotes targeted pollination ofpear crops by honey bees, wherein the composition comprises aformulation comprising the compounds limonene, linalool and α-pinene,diluted in a sugar solution.
 5. The composition according to claim 4,wherein the sugar solution is an aqueous sucrose solution.
 6. Thecomposition according to claim 5, wherein the aqueous sucrose solutionis a 50% w/w aqueous sucrose solution.
 7. The composition according toclaim 5, wherein the composition comprises 0.1 ml to 0.2 ml of theformulation per 1 L of a 50% w/w aqueous sucrose solution.
 8. Thecomposition according to claim 5, wherein the composition comprises 0.1ml to 0.2 ml of a formulation comprising 24% limonene, 35% linalool and41% α-pinene, per 1 L of a 50% w/w aqueous sucrose solution.
 9. A methodfor promoting targeted pollination of pear crops by honey bees, whereinthe method comprises the steps of: a) administering to honey bee hives acomposition that comprises a formulation comprising the compoundslimonene, linalool and α-pinene; b) keeping the hives within or in theimmediate vicinity of the pear crop, the pollination of which is soughtto be promoted; and c) removing the hives.
 10. The method according toclaim 9, wherein step a) is carried out through an artificial feederlocated inside the hive.
 11. The method according to claim 9, whereinstep a) is carried out within 2 days before taking the hives to thecrop, the pollination of which is sought to be promoted.
 12. The methodaccording to claim 9, wherein step a) is carried out after placing thehives in the crop, the pollination of which is sought to be promoted.13. The method according to claim 12, wherein step a) is carried outbefore the crop's blooming level reaches 20%.
 14. The method accordingto claim 9, wherein during step b) a second administration of thecomposition to the hives is carried out.